This R21 application will initiate a series of experiments designed to assess the overall hypothesis that regulated insulin secretion, from primary, reconstituted or ductal cell derived islets, depends on the intra-islet organization, inclusive of coordinated cell function, cell-cell communication and the balance of heterogeneous cell types. The research outlined will test this hypothesis using primary, reconstituted, and engineered islets. This forms the overall aim. We will address this hypothesis by combining new and established procedures for generating reconstituted islets with novel and established experimental procedures to monitor the islet metabolic state, insulin secretion and cellular connectivity within the islets. Our focus will be on the single islet. Data collected from these preparations will offer insights into the critical structural and functional parameters of islets that optimize the secretion of insulin and will provide information on the importance of structural integrity in glucose stimulated insulin secretion (GSIS). The results of this research will also contribute to the promising field of islet transplantation. The application is considered high risk for several reasons: 1. The combined analysis of factors coordinating insulin release in reconstituted or ductal cell derived islets has not been attempted before. 2. Several of the techniques to be used are new to the study of single islets, or still under development. 3. The study of reconstituted islets or islets generated de novo from ductal cells is new to the applicant laboratory. Three specific aims are proposed: Specific Aim 1: What is the time line and sequence of events underlying the emergence of a "normal" islet function on re-association? Specific Aim 2: To what extent do islets with a heterogeneous cell population differ in function to those reformed from a "pure" beta-ceIl population? Specific Aim 3: How do the metabolism, secretion and electrical activity of islets derived from ductal cells conform to the pattern of those derived by reassembly of dissociated primary islets? The data collected from these studies will form the bases for submission of an R01.